An ancient role for CYP73 monooxygenases in phenylpropanoid biosynthesis and embryophyte development

The phenylpropanoid pathway is one of the plant metabolic pathways most prominently linked to the transition to terrestrial life, but its evolution and early functions remain elusive. Here, we show that activity of the t-cinnamic acid 4-hydroxylase (C4H), the first plant-specific step in the pathway, emerged concomitantly with the CYP73 gene family in a common ancestor of embryophytes. Through structural studies, we identify conserved CYP73 residues, including a crucial arginine, that have supported C4H activity since the early stages of its evolution. We further demonstrate that impairing C4H function via CYP73 gene inactivation or inhibitor treatment in three bryophyte species—the moss Physcomitrium patens, the liverwort Marchantia polymorpha and the hornwort Anthoceros agrestis—consistently resulted in a shortage of phenylpropanoids and abnormal plant development. The latter could be rescued in the moss by exogenous supply of p-coumaric acid, the product of C4H. Our findings establish the emergence of the CYP73 gene family as a foundational event in the development of the plant phenylpropanoid pathway, and underscore the deep-rooted function of the C4H enzyme in embryophyte biology.

. Search for CYP73 homologs in Viridiplantae proteomes by reciprocal best hits (RBH).
p. 12 | Appendix Table S2.Quantification of free hydroxycinnamic acids in crude extracts of M. polymorpha and A. agrestis after PA treatment.S3.List of primers and synthesized sequences used in the study.S4.List of multiple reaction monitoring (MRM) methods used for targeted analysis by UHPLC-MS/MS.S5.List of quantification masses and molar response factors used for quantitative analysis of cuticular monomers by GC-TOFMS.

Appendix Figure S1. In vitro C4H assay with Klebsormidium nitens kfl00038_0230 recombinant protein.
Representative UHPLC-MS/MS chromatograms showing the absence of p-coumaric acid production from tcinnamic acid (C4H activity) in in vitro assay using microsomes from yeasts transformed with the pYeDP60:kfl00038_0230 plasmid.Negative control assays were performed with microsomes from yeasts transformed with an empty vector.

Appendix Table S1. Search for CYP73 homologs in Viridiplantae proteomes by reciprocal best hits (RBH).
Forward BLASTp search across 20 Viridiplantae species was performed using A. thaliana CYP73A5 protein sequence as query.For each species, the top hit based on the bit-score was used to perform a reverse BLASTp search against the Arabidopsis thaliana proteome.

p. 9 |
Appendix Figure S8.Search for cis-cinnamic acid in C4H-impaired plants.p. 10 | Appendix Figure S9.Molecular characterization of Mpcyp73a1 CRISPR mutants.p. 11 | Appendix Table P450 CO difference spectra of wild-type and R>A mutated CYP73 proteins.Properly folded CYPs exhibit a maximum absorbance at 450 nm when bound to carbon monoxide.CO difference spectra were determined from 20-fold dilution of microsomal preparations.Each absorbance spectrum was normalized according to its maximum (set to 1). .Western-blot analysis of recombinant PpPpCYP73A-3xHA proteins.Microsomes were 10-fold diluted in denaturation buffer (200 mM Tris pH 6.8, 8% SDS, 40% glycerol, 0.1% bromophenol blue and 100 mM DTT) prior to denaturation at 65°C for 5 min.Ten micrograms total protein were loaded on each lane.AppendixFigure S4.Molecular characterization of Physcomitrium patens DCYP73A mutants.(A) Homologous recombination-mediated strategy for PpCYP73A48 and PpCYP73A49 gene disruption.Genomic fragments encompassing the critical heme-binding site were excised with simultaneous insertion of the NPTII (PpCYP73A48) or HPT (PpCYP73A49) selection cassette conferring resistance to geneticin and hygromycin B, respectively.(B) RT-PCR analysis of DPpCYP73A48, DPpCYP73A49 and DPpCYP73A48/PpCYP73A49 mutant lines showing the absence of corresponding transcripts.The primer hybridization sites for RT-PCR are indicated in panel A. RT-PCR analysis of L21 transcripts was used as amplification control.M, MassRuler™ DNA Ladder (ThermoFisher Scientific).